Influenza A virus (IAV) is a major public health threat, responsible for an estimated 250,000 to 500,000 deaths per year, worldwide. IAV continues to circulate in humans despite the existence of a vaccine, due to the exceptional ability of the virus to escape from herd immunity. In addition, morbidity and mortality rates can rise substantially during unpredictable pandemics that occur when a novel zoonotic strain successfully adapts to circulate in humans. Furthering our basic understanding of IAV infection and adaptation is essential to improving both anti-IAV vaccines and our ability to predict and protect against future pandemics. The ability of IAV to modulate the relative activities of the viral glycoproteins, hemagglutinin and neuraminidase (NA), to optimize interactions with human receptors plays a critical role in pandemic emergence. We recently described a novel network of intersegment epistatic gene regulation through which the virus selectively reduced the expression and gene packaging of the viral NA gene during adaptation to transmit in a new host species. In Aim 1 of this proposal, we will use multiple approaches to identify the molecular mechanisms and additional sequence determinants involved in epistatic regulation of viral gene expression across a diverse range of IAV strains. These results will be critical both for improving our sequence-based methods of predicting IAV adaptation, and for rationally modifying the recombinant virus backbones used in vaccine production to improve antigen content and yield. In Aim 2, we will exploit these findings to explore how variation in relative viral gene expression affects immunogenicity in mice and guinea pigs following either vaccination or infection. Together, these studies will dramatically expand our understanding of IAV biology, adaptation, and immunity and lead the way to new strategies for rationally modifying our current vaccines to improve their yield, breadth, and efficacy.